Possible role of NO/NMDA pathway in the autistic-like behaviors induced by maternal separation stress in mice

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder. Maternal separation (MS) stress is an established model of early-life stress associated with autistic-like behaviors. Altered glutamatergic and nitrergic neurotransmissions may contribute to the pathophysiology of ASD. However, the specific mechanisms underlying these alterations and their relationship to MS-induced autistic-like behaviors remain unclear. Addressing this knowledge gap, this study aims to elucidate the involvement of the nitric oxide (NO)/ N-methyl-D-aspartate (NMDA) pathway in MS-induced autistic-like behaviors in mice. This knowledge has the potential to guide future research, potentially leading to the development of targeted interventions or treatments aimed at modulating the NO/NMDA pathway to ameliorate ASD symptoms. Ninety male Naval Medical Research Institute (NMRI) mice were assigned to six groups (n = 15) comprising a control group (treated with saline) and five groups subjected to MS and treated with saline, ketamine, NMDA, L-NAME, and L-arginine. Behavioral tests were conducted, including the three-chamber test, shuttle box, elevated plus-maze, and marble burying test. Gene expression of iNOS, nNOS, and NMDA-R subunits (NR2A and NR2B), along with nitrite levels, was evaluated in the hippocampus. The findings demonstrated that MS induced autistic-like behaviors, accompanied by increased gene expression of iNOS, nNOS, NR2B, NR2A, and elevated nitrite levels in the hippocampus. Modulation of the NO/NMDA pathway with activators and inhibitors altered the effects of MS. These results suggest that the NO/NMDA pathway plays a role in mediating the negative effects of MS and potentially contributes to the development of autistic-like behaviors in maternally separated mice.


Introduction
Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder that is typified by deficits in social communication, repetitive and restricted behaviors, as well as sensory abnormalities [1].The etiology of ASD is complex and multifactorial, involving both genetic and environmental factors [2].Maternal separation (MS) is a commonly used animal model of early-life stress (ELS) [3].This model has been used extensively to investigate the impact of ELS on neurodevelopment, social communication, and behaviors [4].There is growing evidence that ELS including MS is associated with the development of ASD [5,6].
One of the prominent neurobiological changes associated with MS is altered glutamatergic neurotransmission, which has been suggested to contribute to the development of ASD [7,8].Glutamate is the primary stimulatory neurotransmitter in the central nervous system (CNS).It regulates several physiological processes, including learning and memory, neurodevelopment, and neuronal plasticity [9,10].It also acts on various ionotropic and metabotropic receptors, including N-methyl-D-aspartate (NMDA) receptors [11].NMDA receptors (NMDA-Rs) are glutamate-gated ion channels that are widely expressed throughout the brain and are essential for normal synaptic transmission and plasticity [12,13].The NR2 subunit of NMDA-R is essential for NMDAR function and has four different subtypes (NR2A, NR2B, NR2C, and NR2D) encoded by separate genes.NR2A and NR2B subunits are the most abundant in the brain and have a major role in synaptic plasticity [14].Several lines of evidence suggest that NMDA-Rs may contribute to the pathophysiology of ASD [15].For instance, animal models of ASD have also shown abnormalities in NMDAR-mediated neurotransmission and synaptic plasticity [16,17].Previous studies have determined that dysfunction in the NMDA receptors and imbalances in neuronal excitatory and inhibitory synapses is involved in the pathophysiology of ASD.Indicating that dysfunction at excitatory synapses is associated with ASD [18].In this regards it has been determined that the NMDA-R agonist, cycloserine, could ameliorate autistic symptoms in individuals with ASD [19].On the contrary, some evidence demonstrated that elevated NMDA-R function is also involved in ASDs.In this concept, it has been shown that Memantine, an NMDA-R antagonist, improved ASD-related symptoms [20,21].
Nitric oxide (NO) has a major effect on the balance of glutamatergic neurotransmission [22].This molecule is produced through the conversion of L-arginine (L-arg) by the enzyme nitric oxide synthase (NOS), which is present in three different forms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS).[23,24].nNOS and eNOS are constitutively expressed and produce NO under normal physiological conditions, whereas iNOS is induced by various stimuli such as inflammatory cytokines and lipopolysaccharides and produces large amounts of NO [25].NO regulates synaptic plasticity and is involved in the control of various physiological functions, such as neurotransmission.Additionally, it plays a critical part in the regulation of NMDA-R [26].Besides, NO selectively regulates the S-nitrosylation (SNO) of proteins that control glutamate transport and metabolism via the nNOS-dependent pathway.The dysregulation of this process may contribute to the pathogenesis of various neurological disorders, including ASD [27].Previous studies have demonstrated that ELS such as MS increased the level of nitrite, a final product of NO in the brain [28,29].It has been shown that there is a direct association between levels of nitrite in the brain with neurological and neurodevelopmental disorders [30][31][32][33].Pathogenesis of ASD is also assumed to involve a variety of biological flows activated by NO.The excessive NO levels lead to the production of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondrial dysfunction and neuroinflammation as well as oxidative stress which totally deteriorates autistic behaviors [34].The effects of NO in ASD can either be neuroprotective or neurotoxic, and this is determined by the antioxidant capacity and the levels of ROS/RNS associated with oxidative stress [35].Indicating that imbalance in the NO levels is involved in the pathophysiology of ASD, and can worsen the symptoms of this disease [36].Furthermore, various molecular components including iNOS, interferon-gamma (IFN-γ), toll-like receptors 2, 3, and 4 (TLR2, TLR3, TLR4), nuclear factor-kappa B (NF-κB), and Adenosine A2A receptor (A2AR) signaling pathway intricately contribute to the neuroimmune dysfunction, exerting a profound influence on the pathogenesis of ASD.The dysregulation of these key elements disrupts the delicate balance of immune responses within the CNS, leading to aberrant neuroinflammation and neurodevelopmental processes underlying ASD [37][38][39].
In this study, we aimed to investigate the possible involvement of the NO/NMDA pathway in the autistic-like behaviors in a mouse model of MS stress focusing on the probable alterations in the gene expression of iNOS, nNOS, NR2B, and NR2A as well as levels of nitrite in the hippocampus.

Ethics
All experiments described in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press) set forth by the National Institutes of Health (NIH) and were approved by the Ethics Committee of the Shahrekord University of Medical Sciences (Ethics code: IR.SKUMS.REC.1399.256).All efforts were made to minimize animal suffering and to reduce the number of animals used.

Study design
In this study, pregnant Naval Medical Research Institute (NMRI) mice were obtained from the Pasteur Institute located in Tehran, Iran, and kept under standard laboratory conditions, which included maintaining a temperature of 23˚C ± 2, a 12:12 hours light/dark cycle (with lights turned on at 8:00 a.m.), and ad libitum access to food and water.The day of birth was considered postnatal day zero (PND 0).From PND 2 to PND 14, newborn mice were separated from their mothers for three hours every day (between 10:00 a.m. to 1:00 p.m.).The separated pups were then reintroduced to their mother's cage from PND 15 until PND 25.Lastly, seventy-five maternally separated male mice were randomly divided into five groups (n = 15).To do this, the pups of each mouse were accidentally numbered on the 25th day after birth (PND = 25) and were randomly assigned to the experimental groups.Additionally, fifteen NMRI male mice that were not subjected to the MS paradigm were selected as the control group.The control group (Group 1) was given normal saline (1 ml/kg).The second to sixth groups were the maternally separated mice treated with normal saline (1 ml/kg), L-arginine (L-arg), a NO precursor, (50 mg/kg), NG-Nitroarginine methyl ester (L-NAME), a NOS inhibitor, (10 mg/kg), ketamine, an NMDA-R antagonist, (0.5 mg/kg), and N-methyl-D-aspartate (NMDA), an NMDA-R agonist, (150 mg/kg), respectively.All drugs were administered intraperitoneally (i.p.) for 1 week from PND 51-53 until PND 58-60.The behavioral experiments were conducted immediately after the completion of the treatments and were performed between 09:00 a.m. and 05:00 p.m. Finally, the mice were sacrificed under deep anesthesia using diethyl ether, and their hippocampi were removed for molecular analysis [28].Dose and time of administrations were chosen based on previous studies as well as our pilot study [32,33,40,41].By establishing a bilateral alpha is 0.05 and a confidence interval of 90%, 15 mice were considered for each experimental group based on sample size calculation formula [42].It has been determined that animals show low subject to subject variation [43].In order to minimize animal suffering and reduce the number of animals used, based on formula introduced by Charan et al., 5 mice in each group is the acceptable limit and hence can be considered as adequate sample size to see the effect of drug in animal studies [44].Since, behavioral tests impose different levels of stress to animals, in order to minimize the effect of stress reactivity, from 15 mice in each experimental group, 5 mice subjected to the three chamber sociability test, 5 mice subjected to the shuttle box test and 5 mice subjected to the EPM and MBT.Immediately after behavioral tests, mice were anesthetized with diethyl ether and sacrificed by decapitation and hippocampi samples were harvested for biochemical and molecular evaluations.

Behavioral tests
Three-chamber test.To assess social behaviors, we utilized the three-chamber test.A plexiglass box was partitioned into three chambers, including a middle chamber and two side chambers.Throughout the habituation, sociability, and social preference phases, the mice were given the freedom to explore the box.In the habituation phase, mice were placed in the middle chamber for 5 minutes to acclimatize themselves to the surroundings.To perform the sociability phase, two cylindrical wire cages were put in the two side chambers.During the next stage, one wire cage had a same-sex and same-age mouse that had no prior interaction with the subject mice placed inside it (novel mice 1 or social stimulus 1), and the amount of time spent exploring each chamber was measured for 10 minutes.The other wire cage remained empty.The amount of time spent directly interacting with the social stimulus and the empty chamber (non-social stimulus) was recorded.The sociability index (SI) was calculated as (social stimulus 1-non-social stimulus)/ (social stimulus 1 + non-social stimulus).In the social preference stage (SPI), a novel mouse (new mice 2 or social stimulus 2) with characteristics similar to those of the novel mice 1 was placed in another empty wire cage, and the amount of time spent in each chamber was recorded for 10 minutes.The SPI was determined using the following formula: (social stimulus 2-social stimulus 1)/ (social stimulus 2 + social stimulus 1) [45,46].

Elevated plus-maze (EPM).
To evaluate anxiety-related behaviors in mice, the elevated plus-maze (EPM) was employed.The EPM device was constructed with gray plastic and shaped like a plus sign, with two open arms and two closed arms enclosed by walls facing each other.The platform was raised 50 cm above the ground.Prior to the test, the apparatus was meticulously cleaned with 70% ethanol and allowed to dry.In the test, each mouse was individually placed in the center of the maze and faced one of the closed arms for 5 minutes, allowing it to explore the maze.The time spent in each arm, including the open and closed arms, and the number of entries into each arm were recorded.An arm entry was classified as the placement of all four paws into an arm.Increased time spent in closed arms indicated anxiety-like behavior.After each trial, the maze was cleansed with 70% ethanol to reduce any odor cues left by the mice.The test was conducted in a calm environment with controlled lighting conditions to reduce external disturbances [47].
Marble burying test (MBT).The MBT was performed to evaluate repetitive behavior.To perform the test, each mouse was placed into a clean cage containing 20 marbles (arranged in a uniform grid pattern) and allowed to explore for 20 minutes.The number of marbles buried (covered with at least two-thirds of the bedding) was then counted and recorded for each mouse.The test cage was thoroughly cleaned with 70% ethanol between each test to avoid any potential bias [6,45].
Shuttle box test.To evaluate the passive avoidance memory of mice, the shuttle box apparatus was used.This device includes a bright and a dark chamber, which are connected by a door and have separate grid floors for administering electric shocks.The experiment was conducted over four consecutive days.On the first and second days, the mice were given 5 minutes to explore the apparatus.On the third day, the mice were placed in the bright chamber to begin the acquisition test.After a 2-minute adaptation period, an electric shock (1 mA/second) was delivered to the grid floor of the dark chamber.The latency to enter the dark chamber was measured and recorded.On the fourth day, the mice were placed in the bright chamber again, and the interval between entering the bright chamber and entering the dark chamber (up to 60 seconds) was measured.The apparatus was thoroughly cleaned after each trial to eliminate any potential biases or olfactory cues [6].
Biochemical and molecular tests.Nitrite assay.The Griess reaction method was utilized to evaluate nitrite levels in the hippocampus.Initially, the mice were anesthetized with diethyl ether and sacrificed, and the hippocampus was removed and immediately placed into liquid nitrogen.Hippocampus homogenates were prepared, and nitrite concentrations were determined using a colorimetric assay based on the Griess reaction.In brief, 100 μL of samples was loaded into each well and mixed with 100 μL of Griess reagent.The automated plate reader measured the absorbance at 540 nm after ten minutes of incubation at room temperature.The level of nitrite was determined using a standard curve of sodium nitrite (Sigma, USA) and normalized to the weight of each sample [48].
Real-time PCR analysis for the gene expression of nNOS, iNOS, NR2A, and NR2B.In this study, the gene expression of nNOS, iNOS, NR2A, and NR2B in the hippocampus was assessed using Real-Time PCR.The hippocampus tissue was collected and total RNA was extracted using RNX-plus.The RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan).The gene-specific primers and fluorescent probes for nNOS, iNOS, NR2A, and NR2B were designed and optimized.Real-time PCR was performed on the cDNA samples using a light cycler instrument (Roche Diagnostics, Mannheim, Germany) (Takara Bio).The results were analyzed using the 2 -ΔΔCt method to calculate the relative gene expression levels of nNOS, iNOS, NR2A, and NR2B in the hippocampus.The housekeeping gene B2M was used as a reference gene to normalize the gene expression levels [28,49].Primer sequences present in Table 1.

Data analysis
GraphPad PRISM 8 software was used for statistical analysis.Kolmogorov-Smirnov test was applied to evaluate the normal distribution of data.Brown-Forsythe test was used for evaluation of data homogeneity.Analysis showed normal distribution of data with equal variances, thus one-way analysis of variance (ANOVA), a parametric test, followed by Tukey's post hoc test was used for data analysis.Data presented as Mean± S.E.M. Results were deemed statistically significant at p < 0.05.

Behavioral tests
Effects of NO/NMDA mediators on sociability and social preference indexes in the three-chamber test.Results showed that the MS group exhibited a significant reduction in their SI (p<0.001) in comparison to the control group.However, when MS groups were treated with L-NAME, ketamine, and NMDA, their SI improved significantly (p<0.001).In compared to the saline-treated MS mice (Fig 1).Additionally, we observed that SPI was significantly lower in the MS group in compared to the control group (p<0.001).When MS groups  Effects of NO/NMDA mediators on the passive avoidance memory in the shuttle box test.The results showed that there was no notable difference in the initial phase (T1) of the shuttle box test between experimental groups.However, in the subsequent phase of the test (T2) (step-through latency), results showed markedly reduced in the MS group compared to the control group (p<0.01).Interestingly, the administration of ketamine (p<0.05), and L-NAME (p<0.05)significantly increased the T2 compared to the saline-treated MS group (Fig 4).

Biochemical/Molecular analyses
Effects of NO/NMDA mediators on the nitrite levels in the hippocampus.The results indicated a significant increase in nitrite levels in the hippocampus tissue of the MS group compared to the control group (p<0.001).Treatment of MS mice with L-arg (p<0.001) and NMDA (p<0.001) resulted in a significant elevation of nitrite levels compared to the saline-treated MS The gene expression of iNOS significantly increased in the MS group compared to the control group (p<0.01).Treatment of the MS group with L-NAME (p<0.001) and ketamine (p<0.001)significantly decreased, while treatment with L-arg (p<0.01)significantly increased the gene expression of iNOS compared to the saline-treated MS mice.
The gene expression of nNOS significantly increased in the MS group compared to the control group (p<0.001).Treatment of the MS mice with L-NAME (p<0.001) and ketamine (p<0.001)significantly decreased, while administration of L-arg (p<0.01)significantly increased the gene expression of nNOS compared to the saline-treated MS mice.
We found that the gene expression of NR2A significantly increased in the MS group compared to the control group (p<0.05).Treatment of the MS group with ketamine significantly decreased the gene expression of NR2A compared to the saline-treated MS mice (p<0.001).
Results showed that the gene expression of NR2B significantly increased in the MS group compared to the control group (p<0.05).Treatment of the MS group with ketamine significantly decreased the gene expression of NR2B compared to the saline-treated MS mice (p<0.001).Furthermore, treatment of MS animals with NMDA significantly increased the gene expression of NR2B compared to the saline-treated MS mice (p<0.05).

Discussion
The aim of this study was to investigate the potential role of the NO/NMDA pathway in the development of autistic-like behaviors induced by MS in mice.The significant findings of this study revealed that MS is associated with the development of autistic-like behaviors in adult male mice.Specifically, we observed a decrease in SI and SPI in the three-chamber sociability test, an increased number of buried marbles in the MBT, decreased step-through latency (T2) in the shuttle box test, as well as reduced open arm time and open arm entries in the EPM.These behavioral changes were accompanied by an increase in nitrite levels and the expression of iNOS, nNOS, NR2A, and NR2B genes in the hippocampus.Importantly, treatment with NOS/NMDA-R inhibitors (L-NAME and ketamine) attenuated the negative effects of MS on autistic-like behaviors, decreased levels, and suppressed the expression of iNOS, nNOS, NR2A, and NR2B genes in the hippocampus.Conversely, administration of NOS/NMDA-R activators (L-arg and NMDA) worsened the negative effects of MS on behavior and increased nitrite levels and the expression of iNOS, nNOS, NR2A, and NR2B genes in the hippocampus.
An early direct relationship between infant and mother plays a main role in developing the nervous system [50,51].Many previous studies have reported that early life adversities have enduring and long-lasting negative effects on behavioral, psychological, and neurological features in adulthood [29,52,53].It has been demonstrated that maternal separation (MS) stress as a valid adverse experience in early life significantly increased the risk of neurological and psychiatric disorders in adulthood [54,55].MS, with its structural, developmental, neuroendocrine, and neurochemical changes in different areas of the central nervous system, such as the hippocampus causes mood-behavioral disorders.These changes provide the basis for the incidence of neurodegenerative diseases in adulthood [56].Studies on ASD designated defects in brain development as well as structural disturbances [57].Previous evidence showed that experiencing early life difficulties like MS stress could lead to the development of ASD [6,58].MS through changes in neuronal plasticity triggers the development of ASD [59].It has been shown that modulating neurochemical and neurostructural changes in maternally separated animals could ameliorate autistic-like behaviors [60].Moreover, the presence of heightened oxidative stress and a lack of sufficient antioxidant protection can exacerbate autistic-like behaviors.However, it is possible to alleviate these behaviors by boosting neuronal antioxidants [61].Besides, the pivotal role of neuroinflammation in the pathogenesis of ASD has been extensively documented, revealing intriguing insights into the complex etiology of the disorder.A relevant study conducted by Sheikh et al. provides compelling evidence regarding the potential efficacy of Peroxisome proliferator-activated receptor delta (PPARδ) activation in alleviating behavioral dysfunction in an autistic mouse model.Their research demonstrated that PPARδ, known for its anti-inflammatory effects in animal models of neuroinflammatory diseases, exhibited promising therapeutic effects.By activating PPARδ, which is associated with anti-inflammatory properties, Sheikh et al. observed significant improvements in behavioral dysfunction in the mouse model of autism [62].In this regard, in the orbitofrontal cortex of autistic patients, post-mortem analyses have unveiled dysregulated expression of NF-κB, a key transcription factor associated with inflammatory processes.Notably, heightened activity of microglia, the resident immune cells of the central nervous system, has been observed in conjunction with this aberrant NF-κB expression [63].Compellingly, proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IFN-γ, have exhibited significant upregulation in the brains of individuals with autism [64].Concurrently, investigations have revealed that MS stress can elicit neuroinflammatory responses within the brain [65].Consequently, this induced neuroinflammation may serve as one of pivotal triggers for the emergence and progression of autistic-like behaviors in a mouse model of MS stress.
The main symptoms of ASD are defects in social interaction and communication, as well as repetitive and stereotyped behaviors [66,67].In addition, it has been well-determined that autism is associated with anxiety and memory disturbance [68,69].Preclinical studies have determined that ASD is associated with impaired passive avoidance memory in the shuttle box [6,70].In addition, evidence showed that ASD is associated with a decrease in the time and number of entries in the open arms of the EMP, indicating that ASD is accompanied by anxiety-like behaviors [6,45,71].In the case of repetitive behaviors, previous studies have shown that autistic mice buried more marbles than healthy mice, indicating obsessive-compulsive and repetitive behaviors in autistic mice [72,73].Furthermore, preclinical studies have demonstrated that ASD is linked with deficits in social abilities and social interactions in the three-chamber sociability test [74].The findings of the present study showed that experiencing early life stress as MS is associated with autistic-like behaviors in the adult male mice.We found that MS mice have lower SI and SPI in the three-chamber sociability test and buries more marbles in the MBT than control mice.In addition, we observed that MS stress decreased step-through latency (T2) in the shuttle box as well as decreased open arms time and entries in the EPM.Our results are in line with aforementioned studies and indicating that MS stress caused autistic-like behaviors.
Prior studies have established that NO functions as a dual-acting agent in the CNS [75].While excessive amounts of NO can lead to neuronal damage and cell death [76], maintaining physiological levels of NO is crucial for regulating neurological functions [77].The precise role of NO in autism is an active area of research that remains unclear.However, dysregulated NO levels in the brain may contribute to the development of neurological disorders, including autism [35].Elevated NO levels can interact with oxygen to produce reactive nitrogen species (RNS), leading to oxidative stress and lipid peroxidation [78].Moreover, NO plays a critical role in cellular signal transduction through the SNO mechanism [79].Recent studies have suggested that NO and SNO-modified proteins are involved in the pathophysiology of various neurological disorders, including autism spectrum disorder (ASD).A network of SNO-modified proteins may play a functional role in the synaptic vesicle cycle, neurotransmission, and glutamatergic pathway, all of which are implicated in the development of ASD [80].Results of the present study showed that MS stress is associated with an increase in nitrite levels in the hippocampus.Furthermore, we demonstrated a rise in the gene expression of iNOS and nNOS in the hippocampus of MS mice.Administration of the NOS inhibitor, L-NAME, to the MS mice significantly decreased levels of nitrite and also decrease the gene expression of iNOS and nNOS in the hippocampus.While administration of NO precursor, L-arg, to the MS mice increased levels of nitrite and the gene expression of iNOS and nNOS.In the case of behavioral assessments, we observed that administration of L-NAME increased SI and SPI in the threechamber sociability test and decreased the number of buried marbles in the MBT as well as increased step-through latency (T2), in the shuttle box and increased open arms time and entries in the EPM.While treatment of MS mice with L-arg increased levels of nitrite as well as gene expression of iNOS and nNOS in the hippocampus and also worsen the negative effects of MS on autistic-like behaviors.Considering the bilateral role of NO and NMDA in regulating each other [32], we found that the administration of ketamine, an NMDA-R antagonist, decreased the expression of iNOS and nNOS genes.In this concept, Alqahtani and colleagues demonstrated that ketamine is able to reduce iNOS gene expression in mice with traumatic brain injury [81].Our finding is in line with the abovementioned studies that determined the role of NO dysregulation in ASD.These results, partially at least, indicated that the NO pathway may mediate the development of autistic-like behaviors in the MS stress model in mice.
Previous research has shown that the function of NMDA-R plays a critical role in the regulation of social behavior [82].In both human and animal models, dysfunction of NMDA-R at excitatory synapses has been associated with significant autistic-like manifestations [18].It has been determined that NMDA-R agonists could ameliorate autistic symptoms [19].In this regard, Won et al., have shown that enhancing NMDA-R function can improve autistic-like behavior in genetically mutated mice (Shank2-mutant) [83].On the contrary, some evidence demonstrated that elevated NMDAR function is also involved in ASDs and administration of NMDA-R antagonists improved ASD-related symptoms [20,21].Several previous studies have shown that MS increased the gene expression of NMDA-R subunits (NR2A and NR2B) in the brain [84,85].In agreement with the aforementioned previous studies, in this study, we found that MS is associated with an increase in the gene expression of NR2A and NR2B in the hippocampus.Administration of the NMDA-R antagonist, ketamine, to the MS mice significantly decrease the gene expression of NR2A and NR2B in the hippocampus.While administration of NMDA-R agonist, NMDA, to the MS mice increased levels of nitrite and the gene expression of NR2B in the hippocampus.In the case of behavioral tests, we observed that administration of ketamine increased SI and SPI in the three-chamber sociability test and decreased the number of buried marbles in the MBT as well as increased step-through latency (T2) in the shuttle box, and increased open arms time and entries in the EPM.
Results of this study showed that autistic-like behaviors induced by the MS stress paradigm are due to, partially at least, dysregulation of the NO/NMDA pathway in the hippocampus.
One of the limitations of this study is that the molecular mechanisms underlying the effects of NO/NMDA mediators evaluated on the gene expression level and it therefore requires further investigations to evaluate these effects on the protein levels (using western-blotting, IHC or ELISA).Another limitation of this study is that we did not examine the effects of NO/ NMDA mediators in autistic-like behaviors following MS in female mice.

Conclusions
In conclusion, the findings of this study provide evidence supporting the involvement of the NO/NMDA pathway in the development of autistic-like behaviors triggered by MS stress in adult male mice.Our results suggest that the dysregulation of this pathway in the hippocampus contributes, at least partially, to the manifestation of autistic-like behaviors observed in this model.

Fig 1 .Fig 2 .
Fig 1.The effect of MS stress and NO/NMDA mediators on sociability index and social preference index.The values were calculated for a sample of 5 mice and reported as the mean ± S.E.M.The statistical analysis employed a one-way ANOVA followed by Tukey's post-test.***p<0.001compared to the control group and ### p<0.001 compared to the saline-treated MS group.https://doi.org/10.1371/journal.pone.0292631.g001

Fig 3 .Fig 4 .
Fig 3.The effect of MS stress and NO/NMDA mediators on the open arms entries and time spent in open arms in the EPM.Values were calculated for a sample of 5 mice and reported as the mean ± S.E.M.The statistical analysis employed a one-way ANOVA followed by Tukey's post-test.*p<0.05 and ***p<0.001 in comparison to the control group, # p<0.05, ## p<0.01 and ### p<0.001 in comparison to the saline-treated MS group.https://doi.org/10.1371/journal.pone.0292631.g003

Fig 5 .
Fig 5.The effect of MS stress and NO/NMDA mediators on the nitrite levels in the hippocampus.Values were calculated for a sample of 5 mice and reported as the mean ± S.E.M.The statistical analysis employed a one-way ANOVA followed by Tukey's post-test.***p<0.001compared to the control group, ## p<0.01 and ### p<0.001 in compared to the saline-treated MS group.https://doi.org/10.1371/journal.pone.0292631.g005

Fig 6 .
Fig 6.The effect of MS stress and NO/NMDA mediators on the gene expression of iNOS, nNOS, NR2A, and NR2B in the hippocampus.Values were calculated for a sample of 5 mice and reported as the mean ± S.E.M.The statistical analysis employed one-way ANOVA followed by Tukey's post-test.*p<0.05 and ***p<0.001 in comparison to the control group, # p<0.05, ## p<0.01 and ### p<0.001 in comparison to the saline-treated MS group.https://doi.org/10.1371/journal.pone.0292631.g006